EP2333602B1

EP2333602B1 - Spherical aberration correction for non-descanned applications

Info

Publication number: EP2333602B1
Application number: EP10015589.4A
Authority: EP
Inventors: Glen Ivan Redford
Original assignee: Intelligent Imaging Innovations Inc
Current assignee: Intelligent Imaging Innovations Inc
Priority date: 2009-12-14
Filing date: 2010-12-14
Publication date: 2022-07-20
Anticipated expiration: 2030-12-14
Also published as: US20140126047A1; EP2333602A1; US8634131B2; US20110141558A1; US9195041B2

Description

FIELD

The present invention generally relates to spherical aberration correction in optical microscopes. More specifically, the present invention relates to a device, method and use for spherical aberration correction for non-descanned detection. Even more specifically, an exemplary embodiment of the invention relates to a spherical aberration correction device combined with a multi-photon imaging system.

BACKGROUND

High-power optical microscopes can use objectives with large numerical apertures (NA) resulting in high resolution imaging. In the case of oil or water immersion objectives these NAs can exceed 1.0. In any optical system, the first aberration that causes loss of signal and resolution is spherical aberration. Spherical aberration is the artifact in an imaging system caused by the inability of the optical system to focus axial and off-axis light from a point source to a single point. Modern objectives are highly corrected for spherical aberration, using multiple lens elements to eliminate the effect. However, these objectives are corrected only for a single ideal situation (e.g., for a specific cover glass thickness for samples on the cover glass). In practice, moving deeper into the sample introduces spherical aberration and is usually the limiting factor in deep imaging with these objectives. This is true especially in the case of multi-photon microscopy, which is intended for deep imaging, the sample of interest is rarely in the objective's ideal location.
US 2005/0207003 A1 relates to a microscope with a wavefront modulator for correcting spherical aberrations and an optical relay for controlling a beam diameter from an objective lens.
US2003/0063529 A1 relates to an optical head for a disk drive, wherein variation of the thickness of a protective disc layer is considered by shifting lenses in an optical relay.
US 2003/0063379 A1 relates to a scanning optical microscope with a wavefront converting element for correcting spherical aberration. Examples given for the wavefront converting element are liquid crystal cells and membrane mirrors.
US 2008/0151397 A1 is related to an optical pickup for use in an optical disc device. The optical pickup comprises a spherical aberration means which is formed by an optical lens made of glass or transparent plastic and a mechanism which moves the optical lens in an optical axis direction of the lens.
US 2006/098213 A1 relates to an optical device to reduce the generation of spherical aberration, in which a spherical-aberration correction element disposed within the device behind the objective may be moved along an optical axis.
EP 1 717 623 A1 relates to a laser condensing optical system which condenses laser light in different sections of a medium. In particular, the position of a light source can be changed while ensuring that the intensity and the intensity distribution of light which is incident on a pupil face of the optical system remain constant.
EP 0 859 259 A2 is directed to an adapter for correcting spherical aberrations of objective lens. The adapter is attached to the objective lens imagewise or inserted between a first and second objective lens.
JP 2005 316068 A relates to an optical system capable of changing a light source position while keeping a light quantity and a light quantity distribution incident on a pupil plane of the optical system constant.
JP 2001 264637 A is directed to an optical system for compensating spherical aberration. The optical system is disposed on the image side of the objective lens.

SUMMARY

Object of the present invention is to provide a spherical aberration correction device, a method and a use allowing optimized aberration correction in case of scanning microscopes, in particular for multi-photon imaging.
The above object is achieved by a spherical aberration correction device according to claim 1, by a method according to claim 13 or by a use according to claim 15. Preferred embodiments are subject of the subclaims.
A common optical microscope configuration when simplified includes an infinity objective and a tube lens. The sample is imaged a specific distance from the objective and at the back focal length of the tube lens the magnified image is created.
This image could be recorded by a camera or other imaging device, but in the case of non-descanned detection, the whole signal is combined and measured as a single scalar. For creating an image, the angle of the illumination beam on the back aperture of the objective is changed so that the focal point is also changed. Most objectives are made so that points off the axis and in the focal plane are correctly relayed to the plane of the image. Points out of the focal plane (above or below) are also relayed and measured by the non-descanned detector. In the case of a multi-photon instrument, it is known that the entire signal comes from the focal point. By changing the convergence of the illumination beam on the back aperture of the objective, the focal point can be moved up or down along the optical axis.
Because points above and below the ideal plane (that created by a parallel illumination at the back aperture of the objective) are no longer at the specified ideal location for the objective, spherical aberration will be introduced for these points. Consecutively worse spherical aberration will occur as the focus is moved away from the "ideal" image plane. Spherical aberration has a "direction" to it in that the point-spread-function (PSF) is distorted along the z-axis in one direction or another. Thus one can assign a positive and negative spherical aberration of varying degree.
When the sample being imaged is no longer at the specified ideal situation for the given objective, the image of the "ideal" focal plane will have spherical aberration. The true ideal focal plane (with no spherical aberration) will be located above or below the original plane. As the sample moves further from the ideal condition, the spherical aberration free image will move further from the original plane. For any of these conditions, one should be able to move the focal point to the plane that represents the spherical aberration free focal plane. Thus by changing the convergence of the illumination light, one could correct for spherical aberration across a broad range of samples and imaging conditions. In most situations a variable optical relay can be employed to change the plane of the image volume that is recorded.
Accordingly, one exemplary embodiment of the invention is directed toward a moving optical relay system for selecting a plane of interest from the sample. If this relay were to be motorized as well as the focus of the microscope, one could in an automated fashion correct for the spherical aberration in the current sample plane. Typically this would involve software to select the position of the relay based on the quality of the image.
Primarily, there are two sources of spherical aberration in a given sample, The first is due to the conditions of the sample - usually variations in the thickness of the cover glass. This source of aberration is usually invariant over the sample, but is very hard to predict. To correct for this, the sample must be directly measured and the best plane of interest of the image volume chosen based on the measurement of the sample at several planes. The second source of aberration is due to imaging deeper into the sample. The spherical aberration varies with depth for any given sample medium for any given objective in a known manner. An algorithm can be developed that will give the plane of the image volume needed for any given depth of the sample. The end result is that one has a curve that represents the change in plane location vs. sample depth and a random offset to this curve caused by the sample. The offset can be found empirically and then any depth in the sample can be corrected using the calculated curve.
In the case of multi-photon, spherical aberration directly affects the PSF of the illumination which has a large effect on the multi-photon PSF. Often loss of signal and resolution when imaging deep is attributed to scattering, when for most samples it is due to spherical aberration. When corrected for spherical aberration, a multi-photon system would be able to image deeper and have better signal and resolution.
There are many optical ways that one could take the illumination beam which is usually a laser for multi-photon instruments which is collimated and change its divergence as it hits the back aperture of the objective. This optical system would have to be coupled to the scanner if it exists and usually would also involve the tube lens of the microscope.
One exemplary variable relay system involves two positive lenses. The first lens is placed the distance of its focal length from the image plane. By moving this lens, the desired image plane can be selected. The second lens is placed its focal length from the detector. The image plane that is the focal length of the first lens away will be relayed at infinity to the second lens which images it to an image plane. In this way, the image plane can be fixed in its location, while only one lens element needs to be moved. For scanning based systems, this image plane is then the image plane created by the scan lens. Ideally the distance between the two lenses is equal to the sum of their focal lengths. As the first lens moves, this ideal situation will change, with the primary consequence being that the second lens must be of greater diameter. If positioned correctly and if the two lenses are equal, a "zero" point can be established where the original focal plane is created with no additional magnification or distortion.
In accordance with another exemplary embodiment, one could use a fast, accurate linear motion device to move the first lens. This would allow the possible application where the position of the lens is continuously varied as the imaging condition varies (usually by moving deeper in the sample). For example this could allow three-dimensional imaging where each plane in z is individually corrected for spherical aberration. In practice such a device would allow for deeper imaging into a sample.
An exemplary aspect of the invention is directed toward an apparatus comprising:

a non-descanned optical instrument such as a multi-photon microscope; and
a variable optical system for changing the convergence of the illumination beam on the back aperture of the objective.

This apparatus when combined would provide a way to correct for spherical aberration in an automated fashion.
This device has at least one exemplary advantage that when it is in the "zero" position it effectively has no effect on the image - as if the device were not present, This allows the microscope to be used in a normal fashion when spherical aberration correction is not desired or needed. Because this device can also be made with fast motion control, it will allow for spherical aberration correction without affecting the system performance.
Aspects of the invention are thus directed toward spherical aberration correction in optical microscopes.
Still further aspects of the invention are directed toward spherical aberration correction for non-descanned detection. Non-descanned detection or detectors relate in the sense of the present invention preferably to one or more of a variety of PMT's (Photomultipliers) and/or other detectors available for applications ranging from 2-Photon imaging to time correlated single photon counting (TCSPC) analysis.
Even further aspects of the invention are directed toward a spherical aberration correction device combined with a multi-photon imaging system.
Still further aspects of the invention are directed toward a motorized lens system for selecting the convergence of the illumination beam on the back aperture of the objective.
Even further aspects of the invention are directed toward a fast linear motion device such that the desired motion can happen in less than the retrace time for a scanner system.
Even further aspects of the invention are directed toward automated control and software for the device.
Still further aspects of the invention relate to an apparatus for a spherical aberration correction system for non-descanned detection including:

an optical imaging system such as a microscope for non-descanned detection such as multi-photon; and
an optical system for selecting the convergence of the illumination beam on the back aperture of the objective.

The aspects above, where the optical system is motorized.
The aspects above, where the optical system is motorized by using a moving coil actuator.
The aspects above, where the optical system is motorized using a stepper-motor.
The aspects above, where the optical system is moved using a manual focusing device.
The aspects above, where the motorization control device is synchronized with a scanner and/or a detector.
The aspects above, where the means for moving the optics can do so in under the retrace time of the scanning.
The aspects above, where the imaging system is an optical microscope.
The aspects above, where the apparatus is combined with an electronic imaging device such as a camera.
The aspects above, where the apparatus is combined with a scanning microscope.
The aspects above, where the scanning microscope is a confocal microscope.
The aspects above, where the scanning microscope is a multi-photon microscope.
The aspects above, where the apparatus has a "zero" mode where the effective image is unaltered from the image were the apparatus not present.
The aspects above, where the apparatus is automated and controlled with a computer program.
The aspects above, where the computer program uses a calculated curve to determine the position of the first lens for a given sample depth.
These and other features and advantages of this invention are described and, or are apparent from, the following detailed description of the exemplary embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

The exemplary embodiments of the invention will be described in detail, with reference to the following figures wherein:

Figure 1: illustrates an exemplary optical system for correcting spherical aberration by modifying the illumination path.
Figure 2: illustrates an exemplary embodiment of the device as illustrated in Figure 1.

DETAILED DESCRIPTION

The exemplary embodiments of this invention will be described in relation to microscopes, imaging systems, and associated components. However, it should be appreciated that, in general, known components will not be described in detail. For purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the present invention. It should be appreciated however that the present invention may be practiced in a variety of ways beyond the specific details set forth herein.
The present invention relates to a scanning microscope.
Figure 1 illustrates an exemplary optical system 1 for modifying the illumination path to correct for spherical aberration according to the present invention, The optical system 1, in addition to well known componentry and motors/controllers, comprises a focal plane 10, a front lens 20, a back or moveable lens 30, a focal plane 50, a tube lens 60, an objective 70 and/or a focal point 80, in particular in this sequence
In operation, a scan lens or the like creates a focal plane 10 or light is coming from a scanning device or system (not shown) and the light 5 (preferably illumination or excitation light) is collected by the front lens 20 of the optical system 1. Preferably, an infinity conjugate relay 25 comprises or is formed by the lens 20 and the moveable lens 30.
The light is then collimated and reaches the back or moveable lens 30, which can move along the optical axis 40 with the assistance of a motor/controller module (not shown). The focal plane 50 of the tube lens 60 can then be before or after the focal point from the moving lens 30. The result is that the convergence of the light when the light hits the back aperture of the objective 70 is or can be controlled. For example, as the light changes from convergent to divergent, the focal point 80 moves further away from the objective or vice versa. In this manner, the position of the focal point 80 along the optical axis of the light can be controlled with motion of the lens 30.
The infinity conjugate relay 25 forms preferably a variable optical relay or transmission means that is used to change the divergence/convergence of a light beam and/or to change the plane of an image volume that can be studied or recorded or is in focus. By moving the lens 30, the desired image plane can be selected or changed. In particular, only one lens 30 has to be moved. Ideally, the lens 30 can be moved in a position or at a "zero" point where the original focal plane is imaged with no additional magnification or distortion. However, the conjugate relay 25 or movable lens 30 can be used for other purposes alternatively or addtionally.
Figure 2 illustrates an exemplary environmental perspective view of the embodiment of the optical system as shown in Fig. 1. The optical system includes an enclosure 200 housing a port 210, a moving lens assembly 220 and a port 230. This optical system or moving lens assembly 220 includes the movable lens 30, the stationary and moving lenses 20 and 30, the infinity conjugate relay 25 and/or the optical system 1 as described above. The input illumination beam or light enters through port 210. The beam or light is collimated and hits the moving lens assembly 220. Afterwards, the light passes preferably through the tube lens 60 and objective 80 which are arranged preferably within the enclosure 200 as well, Then the light exits the system or enclosure 200 at the back port 230. This optical system can be directly attached to an entrance port of a microscope (not shown), between, for example, a scanning system and the microscope via well known attachment mechanism(s).
The scanning system can be in accordance with an exemplary embodiment of the invention an optical scanning system such as a laser scanning system and even more particularly a modular laser scanning system. An exemplary laser scanning system is a module with various inner and outer optic modules that provide a complete laser scanning attachment for a microscope. The modular aspect will allow the addition of laser scanning techniques to the already available techniques on the microscope. Importantly, the laser scanning system and software that controls the system can be designed to work seamlessly with the microscope.
Preferably, the scanning system illuminates and/or scans with a light beam, in particular a laser beam, a sample which is studied by the optical imaging system, such as a microscope.
Preferably, the scanning system and the optical imaging system cooperate or the sanning system may form part of the optical imaging system. The optical system is preferably arranged between the scanning system and the optical imaging system.
The exemplary techniques illustrated herein are not limited to the specifically illustrated embodiments but can also be utilized with the other exemplary embodiments and each described feature is individually and separately claimable.
The systems of this invention also can cooperate and interface with a special purpose computer, a programmed microprocessor or microcontroller and peripheral integrated circuit element(s), an ASIC or other integrated circuit, a digital signal processor, a hard-wired electronic or logic circuit such as discrete element circuit, a programmable logic device such as PLD, PLA, FPGA, PAL, any comparable means, or the like. The term module as used herein can refer to any known or later developed hardware, software, firmware, or combination thereof, that is capable of performing the functionality associated with that element. The terms determine, calculate, and compute and variations thereof, as used herein are used interchangeable and include any type of methodology, process, technique, mathematical operational or protocol.
Furthermore, the disclosed system may use control methods/systems and graphical user interfaces that may be readily implemented in software/hardware using object or object-oriented software development environments that provide portable source code that can be used on a variety of computer or workstation platforms that include a processor and memory. Alternatively, the disclosed control methods may be implemented partially or fully in hardware using standard logic circuits or VLSI design. Whether software or hardware is used to implement the systems in accordance with this invention is dependent on the speed and/or efficiency requirements of the system, the particular function, and the particular software or hardware systems or microprocessor or microcomputer systems being utilized.
It is therefore apparent that there has been provided, in accordance with the present invention an optical system, While this invention has been described in conjunction with a number of embodiments, it is evident that many alternatives, modifications and variations would be or are apparent to those of ordinary skill in the applicable arts.

Claims

A spherical aberration correction device for non-descanned detection or detectors, comprising:
a scanning microscope, preferably for multi-photon imaging, adapted for non-descanned detection; and

an optical system including an enclosure (200), the enclosure (200) housing a first port (210), a second port (230) and a moving lens assembly (220) including a moveable lens (30),

wherein the optical system is adapted to select a convergence of illumination light (5) on a back aperture of an objective (70), wherein, as the light (5) changes from convergent to divergent, a focal point (80) moves further away from the objective (70) or vice versa, such that the convergence of illumination light (5) corrects spherical aberration,

wherein the light (5) is collimated and reaches the moveable lens (30), which is configured to move along its optical axis (40), such that the position of the focal point (80) along the optical axis (40) of the light (5) can be controlled with motion of the lens (30), and

wherein the optical system is directly attached via the second port (230) to an entrance port of the scanning microscope and the input illumination beam enters the optical system through the first port (210).
Device of claim 1, wherein the optical system (1) is adapted such that the light (5) passes through a tube lens (60) and the objective (70), wherein, by moving the movable lens (30), a focal plane (50) of the tube lens (60) can be before or after the focal point from the moving lens (30).
Device of claim 2, wherein resulting data is not processed for imaging, but is processed to provide other information about a sample including one or more of flow rates and diffusion.
Device according to any one of the preceding claims, where the optical system is a variable optical relay, preferably wherein the optical relay is an infinity- conjugate relay.
Device according to any one of the preceding claims, where the optical system is motorized.
Device of claim5, wherein the optical system can move during a retrace time of a scanner or during imaging.
Device of claim 5 or 6, where the optical system (1) is controlled electronically by a computer and/or an electronic device preferably wherein the electronic device adjusts for spherical aberration based only on image data.
Device of claim 7, wherein software uses a stored equation or numerical formula to adjust the spherical aberration correction for any given sample depth, and/or wherein the software automatically adjusts the correction during live imaging and/or uses an empirically derived offset to the correction to account for sample conditions.
Device according to any one of the preceding claims, wherein spherical aberration is corrected for each plane individually in a three-dimensional data set.
Device according to any one of the preceding claims, wherein the optical imaging system (1) is not used for imaging and/or detects a signal from a single point, line or other arbitrary shape.
Device according to any one of the preceding claims, wherein the optical system (1) comprises an enclosure housing, a front lens (20), a movable back lens (30), a tube lens (60), and an objective (70), in particular in this sequence, wherein light (5) collected by the front lens (20) from a scan lens is collimated until it reaches the movable back lens (30), the back lens (30) being movable along an optical axis (40), and preferably controllable based on a stored equation or numerical formula, to adjust spherical aberration correction, with the tube lens (60) capable of converging the light (5) when the light (5) encounters a back aperture of the objective (70).
Method for spherical aberration correction with a spherical aberration correction device for non-descanned detection or detectors, comprising:
a scanning microscope, preferably for multi-photon imaging, adapted for non-descanned detection; and

an optical system including an enclosure (200), the enclosure (200) housing a first port (210), a second port (230) and a moving lens assembly (220) including a moveable lens (30),

wherein the optical system selects a convergence of illumination light (5) on a back aperture of an objective (70), wherein, as the illumination light (5) changes from convergent to divergent, a focal point (80) moves further away from the objective (70) or vice versa, such that the convergence of illumination light (5) corrects spherical aberration,

wherein light collected by a front lens from a scan lens is collimated until it reaches the movable lens, wherein the moveable lens is moved along an optical axis and controlled based on a stored equation or numerical formula to adjust spherical aberration correction,

wherein a tube lens converges the light when the light encounters a back aperture of an objective, and

wherein the optical system is directly attached via the second port (230) to an entrance port of the scanning microscope and the input illumination beam enters the optical system through the first port (210).
Use of an optical system including an enclosure (200), the enclosure (200) housing a first port (210), a second port (230) and a moving lens assembly (220) including a moveable lens (30), wherein the optical system (1) selects a convergence of illumination light (5) on a back aperture of an objective (70), wherein, as the illumination light (5) changes from convergent to divergent, a focal point (80) moves further away from the objective (70) or vice versa, such that the convergence of illumination light (5) corrects spherical aberration, in a scanning microscope, preferably for multi-photon imaging, wherein the light (5) is collimated and reaches the moveable lens (30), which can move along the optical axis (40), such that the position of the focal point (80) along an optical axis (40) of the light (5) can be controlled with motion of the lens (30), and wherein the optical system is directly attached via the second port (230) to an entrance port of the scanning microscope and the input illumination beam enters the optical system through the first port (210).